Monobodies are small recombinant proteins designed to bind with high affinity to target proteins. Monobodies have been generated to mimic the SIM [SUMO (small ubiquitin-like modifier)-interacting motif] present in many SUMO target proteins, but their properties have not been determined in cells. In the present study we characterize the properties of two SUMO1-specific monobodies (hS1MB4 and hS1MB5) in HEK (human embyronic kidney)-293 and HeLa cells and examine their ability to purify SUMO substrates from cell lines and rat brain. Both hS1MB4 and hS1MB5 compared favourably with commercially available antibodies and were highly selective for binding to SUMO1 over SUMO2/3 in pull-down assays against endogenous and overexpressed SUMO and SUMOylated proteins. Monobodies expressed in HeLa cells displayed a nuclear and cytosolic distribution that overlaps with SUMO1. Expression of the monobodies effectively inhibited protein SUMOylation by SUMO1 and, surprisingly, by SUMO2/3, but were not cytotoxic for at least 36 h. We attribute the effects on SUMO2/3 to the role of SUMO1 in chain termination and/or monobody inhibition of the SUMO-conjugating E1 enzyme complex. Taken together, these data provide the first demonstration that monobodies represent useful new tools both to isolate SUMO conjugates and to probe cell SUMOylation pathways in vivo.